Uses of antibodies to interleukin-6 in cancer treatment

ABSTRACT

The present disclosure provides antibodies that bind to human interleukin-6 (IL6). The antibodies can modulate IL6 signaling and thus used in treatment or prevention of IL6 associated diseases or disorders, particularly inflammatory disorder, rheumatoid arthritis (RA), angiogenesis, and cancer.

RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.14/985,642, filed on Dec. 31, 2015, which is a divisional application ofU.S. Ser. No. 14/057,349, filed on Oct. 18, 2013, which claims thebenefit of U.S. provisional application No. 61/716,802, filed Oct. 22,2012 under 35 U.S.C. §119, the entire content of each of which isincorporated by reference herein.

BACKGROUND OF THE INVENTION

Human interleukin-6 (IL6), a secreted glycoprotein having 184 aminoacids (21 kDa), has a four-helix bundle structure. IL6 is amulti-functional cytokine that acts on various type of cells, e.g., Bcells, T cells, fibroblasts, hepatocytes, osteoclasts, neural cells,mesangial cells, epidermal keratinocytes, and hematopoietic progenitorcells, via binding to two distinct receptor proteins, the IL6 receptor(IL6R) and glycoprotein 130 (gp130). Formation of the IL6/IL6R/gp130complex transduces intracellular signaling pathways, including thosemediated by (1) phosphatidyl inositol-3′-kinase (PI3K), (2)mitogen-activated protein kinase (MAK), and (3) Janus tyrosine kinase(JAK)-signal transducer and activator of transcription 1 and 3 (STAT1and STAT3).

IL6 functions as an immune regulator, cell growth factor, bonemetabolism regulator, cell differentiation factor, and acute phaseprotein inducer against several effecter cells. In liver, IL6 inducesvarious acute-phase proteins such as serum amyloid A (SAA), C-reactiveprotein (CRP), hepcidin, fibrinogen, and haptoglobin antichymotrypsin.The pathological significance of IL6 for various diseases has beenindicated in numerous studies, including chronic inflammatory diseases,autoimmune diseases (e.g., rheumatoid arthritis, Crohn's disease,Castleman's disease and psoriasis), cancer (e.g., multiple myeloma,leukemia, breast cancer, pancreatic cancer, prostate cancer and variouscancers), and cachexia and coronary heart disease.

It is therefore of great interest to develop new IL6 antagonists for usein treating diseases associated with the IL6 signaling.

SUMMARY OF THE INVENTION

The present disclosure is based on the identification of a number ofexemplary anti-IL6 antibodies, e.g., 1-4-62, Ag1-4-6 (also known asFB704), or HAg1T-3-10, which unexpectedly showed high binding affinityand specificity to human IL6, and superior activities in inhibitingIL6-induced cell proliferation (e.g., cancer cell proliferation) andcytokine production (e.g., inflammatory cytokine production),angiogenesis, cancer-induced cachexia, and cancer metastasis. Suchantibodies also significantly enhanced anti-cancer effects of otherchemotherapeutic agents such as oxaliplatin, gemcitabine, and docetaxel.

Accordingly, one aspect of the present disclosure relates to an isolatedantibody that binds to human interleukin 6 (IL6), comprising:

(a) a heavy chain variable region (V_(H)), which comprises a heavy chaincomplementary determining region 1 (HC CDR1) of SEQ ID NO: 2, a heavychain complementary determining region 2 (HC CDR2) of SEQ ID NO: 4, anda heavy chain complementary determining region 3 (HC CDR3) of SEQ ID NO:6 or SEQ ID NO: 16; or

(b) a light chain variable region (V_(L)), which comprises a light chaincomplementary determining region 1(LC CDR)1 of SEQ ID NO: 9, a lightchain complementary determining region 2 (LC CDR2) of SEQ ID NO: 11, anda light chain complementary determining region 3 (LC CDR3) of SEQ ID NO:13 or SEQ ID NO: 15.

In some embodiments, the isolated anti-IL6 antibody comprises (i) aV_(H) that comprises the HC CDR1 of SEQ ID NO: 2, the HC CDR2 of SEQ IDNO: 4, and the HC CDR3 of SEQ ID NO: 6; or (ii) a V_(H) that comprisesthe HC CDR1 of SEQ ID NO: 2, the HC CDR2 of SEQ ID NO: 4, and the HCCDR3 of SEQ ID NO: 16. In one example, the antibody comprises a V_(H)that comprises the amino acid sequence of SEQ ID NO:17 or SEQ ID NO:18.

In other embodiments, the isolated anti-IL6 antibody further comprises(i) a V_(L) that comprises the LC CDR1 of SEQ ID NO: 9, the LC CDR2 ofSEQ ID NO: 11, and the LC CDR3 of SEQ ID NO: 13; or (ii) a V_(L) thatcomprises the LC CDR1 of SEQ ID NO: 9, the LC CDR2 of SEQ ID NO: 11, andthe LC CDR3 of SEQ ID NO: 15. In one example, the antibody furthercomprises a V_(L) that comprises the amino acid sequence of SEQ ID NO:19or SEQ ID NO:20.

Examples of the anti-IL6 antibodies as described here include, but arenot limited to:

(i) an antibody comprising a V_(H) that comprises the HC CDR1 of SEQ IDNO: 2, the HC CDR2 of SEQ ID NO: 4, and the HC CDR3 of SEQ ID NO: 6; anda V_(L) that comprises the LC CDR1 of SEQ ID NO: 9, the LC CDR2 of SEQID NO: 11, and the CDR3 of SEQ ID NO: 13;

(ii) an antibody comprising a V_(H) that comprises the HC CDR1 of SEQ IDNO: 2, the HC CDR2 of SEQ ID NO: 4, and the HC CDR3 of SEQ ID NO: 16,and a V_(L) that comprises the LC CDR1 of SEQ ID NO: 9, the LC CDR2 ofSEQ ID NO: 11, and the CDR3 of SEQ ID NO: 13;

(iii) an antibody comprising a V_(H) that comprises the HC CDR1 of SEQID NO: 2, the HC CDR2 of SEQ ID NO: 4, and the HC CDR3 of SEQ ID NO: 6,and a V_(L) that comprises the LC CDR1 of SEQ ID NO: 9, the LC CDR2 ofSEQ ID NO: 11, and the CDR3 of SEQ ID NO: 15;

(iv) an antibody comprising a V_(H) that comprises the HC CDR1 of SEQ IDNO: 2, the HC CDR2 of SEQ ID NO: 4, and the HC CDR3 of SEQ ID NO: 16,and a V_(L) that comprises the LC CDR1 of SEQ ID NO: 9, the LC CDR2 ofSEQ ID NO: 11, and the CDR3 of SEQ ID NO: 15;

(v) an antibody comprising a V_(H) that comprises the amino acidsequence of SEQ ID NO: 17 and a V_(L) that comprises the amino acidsequence of SEQ ID NO:19;

(vi) an antibody comprising a V_(H) that comprises the amino acidsequence of SEQ ID NO: 17 and a V_(L) that comprises the amino acidsequence of SEQ ID NO:20; and

(vii) an antibody comprising a V_(H) that comprises the amino acidsequence of SEQ ID NO: 18 and a V_(L) that comprises the amino acidsequence of SEQ ID NO:19; and

(viii) an antibody comprising a V_(H) that comprises the amino acidsequence of SEQ ID NO: 18 and a V_(L) that comprises the amino acidsequence of SEQ ID NO:20.

Any of the anti-IL6 antibodies described herein can be a full-lengthantibody or an antigen-binding fragment thereof, which can be Fab or(Fab′)₂. Alternatively, the anti-IL6 antibody can be a single chainantibody, a humanized antibody, or a human antibody.

In another aspect, the present disclosure provides a nucleic acidcomprising a nucleotide sequence encoding an antibody heavy chainvariable region (V_(H)), an antibody light chain variable region (V_(L))or both, wherein the V_(H) and V_(L) are as described herein.

In another aspect, the present disclosure provides a vector (e.g., anexpression vector) comprising any of the nucleic acids described hereinand a host cell comprising such a vector.

In yet another aspect, the present disclosure provides a method forproducing an antibody that binds to human IL6, comprising: (i) culturingthe host cell as described herein under conditions allowing forexpression of the antibody, and optionally, (ii) harvesting theantibody.

Another aspect of the present disclosure relates to a composition (e.g.,a pharmaceutical composition) comprising (a) any of the anti-IL6antibody described herein, any of the nucleic acids described herein, orany of the vectors described herein; and (b) a carrier such as apharmaceutically acceptable carrier.

In some embodiments, any of the compositions described herein furthercomprises another anti-cancer agent or a disease modifying antirheumaticdrug (DMARD). Examples of the anti-cancer agent include, but are notlimited to, docetaxel, oxaliplatin, and gemcitabine. Examples of DMARDsinclude, but are not limited to, methotrexate, azathioprine, chloroquinehydroxychloroquine, cyclosporin A, and sulfasalazine.

In yet another aspect, the present disclosure provides a method fortreating a disease associated with IL6, comprising administering to asubject in need thereof a therapeutically effective amount of any of theanti-IL6 antibodies described herein, or any of the nucleic acids thatencode such anti-IL6 antibodies. Examples of the disease associated withIL6 include, but are not limited to, inflammatory disorder, autoimmunediseases, angiogenesis, and cancer.

In some embodiments, the disease associated with IL6 is cancer, whichcan be multiple myeloma, leukemia, breast cancer, pancreatic cancer,lung cancer, ovarian cancer, oral cancer and prostate cancer. In someexamples, the amount of the antibody or the encoding nucleic acid iseffective in reducing tumor metastasis or cancer related cachexia. Inother examples, the method further comprises administering to thesubject another anti-cancer agent, e.g., oxaliplatin, gemcitabine, ordocetaxel.

In other embodiments, the disease associated with IL6 is an autoimmunedisease, e.g., rheumatoid arthritis (RA), Crohn's disease, Castleman'sdisease, multiple sclerosis, ankylosing spondylitis, psoriaticarthritis, or psoriasis. In some examples, the method described hereinfurther comprises administering to the subject one or more diseasemodifying antirheumatic drugs (DMARDs), e.g., methotrexate,azathioprine, chloroquine, hydroxychloroquine, cyclosporin A,sulfasalazine.

Also within the scope of the present disclosure are pharmaceuticalcompositions for use in treating a disease associated with IL6, whereinthe pharmaceutical composition comprises (a) any of the anti-IL6antibodies or any of the nucleic acids/vectors described herein; (b) apharmaceutically acceptable carrier; and optionally, (c) an anti-canceragent or a DMARD as those described herein. Exemplary diseasesassociated with IL6 include, but are not limited to, inflammatorydisorder, autoimmune diseases (e.g. rheumatoid arthritis (RA), Crohn'sdisease, Castleman's disease, multiple sclerosis, ankylosingspondylitis, psoriatic arthritis and psoriasis), angiogenesis, cancer(e.g. multiple myeloma, leukemia, breast cancer, pancreatic cancer, lungcancer, ovarian cancer, oral cancer and prostate cancer), tumormetastasis, cancer related cachexia.

Further, the present disclosure provides uses of any of the anti-IL6antibodies or any of the encoding nucleic acids in medicament, or foruse in the manufacture of a medicament for treating a disease orcondition associated with IL6 as those described herein.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following drawings and detaileddescription of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the efficacy of high-affinity anti-IL6antibodies on cell signaling transduction pathway and proliferation. (A)phosphorylated-STAT3 signaling was decreased by anti-IL6 antibodiesAg1-4-6 and HAgT1-3-10 in a dose-dependent manner. (B) Anti-IL6antibodies Ag1-4-6 and HAgT1-3-10 suppressed the STAT3 signaling at amuch greater level as compared to control antibody Actemra. (C) Anti-IL6antibodies 1-4-62, Ag1-4-6 and HAg1T-3-10 inhibited B9 cellproliferation in a dose-dependent manner and the IC₅₀ was 0.618, 0.0468and 0.00456 μg/ml respectively.

FIG. 2 is a diagram showing that anti-IL6 antibodies suppressedchemokine production in HUVEC cells. HUVECs were cultured with IL6,sIL6R, IL6 and sIL6R, or a combination of IL6, sIL6R, and an anti-IL6antibody as indicated for 24 h. MCP-1 and sICAM-1 in the culturesupernatant were measured by ELISA. (A) IL6+sIL6R induced MCP-1secretion was inhibited by anti-IL6 antibodies Ag1-4-6 and Hag1T-3-10.(B) Antibodies Ag1-4-6 and Hag1T-3-10 inhibited production of sICAM-1 ata level greater than Actemra.

FIG. 3 is a diagram showing binding specificity of exemplary anti-IL6antibodies described herein.

FIG. 4 is a diagram showing that antibody Ag1-4-6 (FB704) inhibitedangiogenesis in vivo. hIL6 recombinant protein was added into matrigeland injected in two sites on the dorsal side of mice. Mice were treatedwith antibodies through i.v. injection or pre-mixture. (A) FB704inhibited angiogenesis induced by IL6 as observed 6 days aftertreatment. (B) Treatment with antibody FB704 significantly changed thehemoglobin concentrations.

FIG. 5 is a diagram showing that antibody FB704 inhibited human prostatecancer cell PC-3 induced cachexia and metastasis. After tumor injection,mice body weights were measured on day 31. (A) The body weights ofPBS-treated mice was decreased by approximately 19%. In contrast, highdose FB704- and Actemra-treated groups remained stable and showedsignificant different (P<0.01). (B) FB704 (n=15; P=0.00001) and Actemra(n=12; P=0.024) significantly prolonged symptom-free survival of PC3tumor-bearing mice. FB704 showed significant better efficacy thanActemra (P=0.03). (C) Gross necropsy of PBS treated group showed severeenlargement and tumor cell infiltration in liver. However, FB704 treatedgroup showed moderate tumor cell infiltration and nearly 50% normalhepatocyte in the left and middle lobes of liver. (D)Immunohistochemistry showed the vessel density was decreased after FB704treated on tumor sections. (E) Semi-quantitative analysis of CD31staining showed significantly decreased after FB704 treated (P<0.05).(F) Combination treatment of FB704 plus chemo-drug Docetaxel providedbetter overall survival rate.

FIG. 6 is a diagram showing that antibody FB704 enhanced the anti-tumoractivity of Oxaliplatin or Gemcitabine of pancreatic carcinomaxenograft. (A) Treatment of BxPC-3 tumor-bearing mice with 20 mg/kg ofFB704 twice a week plus Oxaliplatin (3 mg/kg) once a week resulted instatistically significant tumor growth inhibition of 49% (P<0.01). (B)The mouse body weight had normally increased during the treatment. (Cand D) The individual tumor masses were measured after the experimentand the tumor weights were significantly decreased after the treatments.(E) Tumor cell proliferation marker, Ki-67 was showed on differenttreatments of tumor section. (F) Ki-67 positive cell rate of PBS,Oxaliplatin, FB704 and FB704 plus Oxaliplatin treatment groups wereshowed 26%, 14%, 16% and 7.5% respectively. (G) Treatment of BxPC-3tumor-bearing mice with 20 mg/kg of FB704 plus Gemcitabine (80 mg/kg)twice a week resulted in statistically significant tumor growthinhibition of 60% (P<0.01).

FIG. 7 is a diagram showing that exemplary anti-IL6 antibodies describedherein suppressed MCP-1 production on U937 and human PBMC cells. (A)U937 cells were cultured with IL6 and treated by antibodies for 24 h.MCP-1 was measured by ELISA kit. Our antibodies show dose-dependentlysuppression of MCP-1 production (n=3). (B) PBMC cells were cultured withIL6 and treated by antibodies for 24 h. MCP-1 was measured by ELISA. Ourantibodies showed dose-dependently suppression of MCP-1 production(n=5).

FIG. 8 is a diagram showing the Inhibition of MCP-1 production in RA-FLSby exemplary anti-IL6 antibodies described herein. (A) Commercial RA-FLScells and (B) RA patients' FLS cells were cultured with IL6 plus sIL6Rpresent or absent of antibodies for 24 h. Our antibodies showdose-dependently suppression of MCP-1 production (n=6).

FIG. 9 is a diagram showing the inhibition of VEGF production in RA-FLSby exemplary anti-IL6 antibodies described herein. (A) Commercial RA-FLScells and (B) RA patients' FLS cells were cultured with IL6, sIL6R andIL1β and treated with antibodies for 24 h. Our antibodies showdose-dependently suppression of VEGF production (n=6).

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to antibodies that bind humaninterleukin-6 (IL6), which may neutralize IL6 activity, and their usesin regulating IL6-mediated signaling pathways. The anti-IL6 antibodiesdescribed herein are useful in the treatment of IL6 associated diseasesor disorders, such as inflammatory disorders, autoimmune diseases,angiogenesis, cancer, tumor metastasis and cancer related cachexia.

The following description is merely intended to illustrate variousembodiments of the invention. As such, specific embodiments discussedherein are not to be construed as limitations to the scope of theinvention. It will be apparent to one skilled in the art that variouschanges or equivalents may be made without departing from the scope ofthe invention.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook, et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press,Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

Definitions

In order to provide a clear and ready understanding of the presentdisclosure, certain terms are first defined. Additional definitions areset forth throughout the detailed description. Unless defined otherwise,all technical and scientific terms used herein have the same meanings asis commonly understood by one of skill in the pertinent art.

As used herein, the articles “a” and “an” refer to one or more than one(i.e., at least one) of the grammatical object of the article. By way ofexample, “an element” means one element or more than one element.

As used herein, the term “polypeptide” refers to a polymer composed ofamino acid residues linked via peptide bonds. The term “protein”typically refers to relatively large polypeptides. The term “peptide”typically refers to relatively short polypeptides (e.g., containing upto 100, 80, 60, 50, 30, or 20 amino acid residues).

An antibody (interchangeably used in plural form) is an immunoglobulinmolecule capable of specific binding to a target, such as acarbohydrate, polynucleotide, lipid, polypeptide, etc., through at leastone antigen recognition site, located in the variable region of theimmunoglobulin molecule. As used herein, the term “antibody” encompassesnot only intact (i.e., full-length) polyclonal or monoclonal antibodies,but also antigen-binding fragments thereof (such as Fab, Fab′, F(ab′)₂,Fv)), mutants thereof, fusion proteins comprising an antibody portion,humanized antibodies, chimeric antibodies, diabodies, linear antibodies,single chain antibodies, multispecific antibodies (e.g., bispecificantibodies) and any other modified configuration of the immunoglobulinmolecule that comprises an antigen recognition site of the requiredspecificity, including glycosylation variants of antibodies, amino acidsequence variants of antibodies, and covalently modified antibodies.

An intact or full-length antibody comprises two heavy chains and twolight chains. Each heavy chain contains a heavy chain variable region(V_(H)) and a first, second and third constant regions (C_(H)1, C_(H)2and C_(H)3). Each light chain contains a light chain variable region(V_(L)) and a constant region (CL). A full-length antibody can be anantibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-classthereof), and the antibody need not be of any particular class.Depending on the antibody amino acid sequence of the constant domain ofits heavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chainconstant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

The term “antigen-binding domain” or “antigen-binding fragment” refersto a portion or region of an intact antibody molecule that isresponsible for antigen binding. An antigen-binding domain may comprisethe heavy chain variable region (V_(H)), the light chain variable region(V_(L)), or both. Each of the V_(H) and V_(L) typically contains threecomplementarity determining regions CDR1, CDR2, and CDR3. The three CDRsin the V_(H) or V_(L) are franked by framework regions (FR1, FR2, FR3,and FR4).

Examples of antigen-binding fragments of include, but are not limitedto: (1) an Fab fragment, which can be a monovalent fragment having aV_(L)-C_(L) chain and a V_(H)-C_(H)1 chain; (2) an F(ab′)₂ fragment,which can be a bivalent fragment having two Fab fragments linked by adisulfide bridge at the hinge region, i.e. a dimer of Fab; (3) an Fvfragment having the V_(L) and V_(H) domains of a single arm of anantibody; (4) a single chain Fv (scFv), which can be a singlepolypeptide chain composed of a V_(H) domain and a V_(L) domain througha peptide linker; and (5) a (scFv)₂, which can comprise two V_(H)domains linked by a peptide linker and two V_(L) domains, which areassociated with the two V_(H) domains via disulfide bridges.

The term “human antibody” refers to antibodies having variable andconstant regions corresponding substantially to, or derived from,antibodies obtained from human subjects, e.g., encoded by human germlineimmunoglobulin sequences or variants thereof. The human antibodiesdescribed herein may include one or more amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo). Such mutations may present in one or more of the CDRs, or inone or more of the FRs. In some examples, the human antibodies may haveat least one, two, three, four, five, or more positions replaced with anamino acid residue that is not encoded by the human germlineimmunoglobulin sequence.

An “isolated” substance means that it has been altered by the hand ofman from the natural state. If an “isolated” substance presents innature, it has been changed or removed from its original environment, orboth. For example, a polypeptide naturally present in a living subjectis not “isolated” but the polypeptide is isolated if it has beensubstantially separated from the coexisting materials of its naturalstate and exist in a substantially pure state.

The term “specific binds” or “specifically binding” refers to anon-random binding reaction between two molecules, such as the bindingof the antibody to an epitope of the antigen. An antibody that“specifically binds” to a target or an epitope is a term well understoodin the art, and methods to determine such specific binding are also wellknown in the art. A molecule is said to exhibit “specific binding” if itreacts or associates more frequently, more rapidly, with greaterduration and/or with greater affinity with a particular target antigenor an epitope than it does with alternative targets/epitopes. Anantibody “specifically binds” to a target antigen if it binds withgreater affinity, avidity, more readily, and/or with greater durationthan it binds to other substances. For example, an antibody thatspecifically (or preferentially) binds to an IgE epitope is an antibodythat binds this IgE epitope with greater affinity, avidity, morereadily, and/or with greater duration than it binds to other IgEepitopes or non-IgE epitopes. It is also understood by reading thisdefinition that, for example, an antibody that specifically binds to afirst target antigen may or may not specifically or preferentially bindto a second target antigen. As such, “specific binding” or “preferentialbinding” does not necessarily require (although it can include)exclusive binding. Generally, but not necessarily, reference to bindingmeans preferential binding.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein and refer to a mammal being assessed fortreatment and/or being treated. Subjects may be human, but also includeother mammals, particularly those mammals useful as laboratory modelsfor human disease, e.g. mouse, rat, rabbit, dog, etc.

The term “treatment” or “treating” refers to an action, application ortherapy, wherein a subject, including a human being, is subjected tomedical aid with the purpose of improving the subject's condition,directly or indirectly. Particularly, the term refers to reducingincidence, or alleviating symptoms, eliminating recurrence, preventingrecurrence, preventing incidence, improving symptoms, improvingprognosis or combination thereof in some embodiments. The skilledartisan would understand that treatment does not necessarily result inthe complete absence or removal of symptoms. For example, with respectto cancer, “treatment” or “treating” may refer to slowing neoplastic ormalignant cell growth, proliferation, or metastasis, preventing ordelaying the development of neoplastic or malignant cell growth,proliferation, or metastasis, or some combination thereof.

An “effective amount” or an “effective dose” or a “therapeuticallyeffective amount” in connection with administration of a pharmacologicalagent, as used herein, refers to an amount of a drug or pharmaceuticalagent which, as compared to a corresponding subject who has not receivedsuch amount, results in an intended pharmacological result, or an effectin treatment, healing, prevention, or amelioration of a disease,disorder, or side effect, or a decrease in the rate of advancement of adisease or disorder. The effective amount or dose of a pharmacologicalagent may vary depending on particular active ingredient employed, themode of administration, and the age, size, and condition of the subjectto be treated. Precise amounts of a pharmacological agent are requiredto be administered depend on the judgment of the practitioner and arepeculiar to each individual.

An “IL6 associated diseases or conditions” refer to any disease orcondition in which IL6 plays a regulatory role in the signaling pathwayleading to that disease or disorder. IL6 is a member of a family ofcytokines that initiate cellular responses through a receptor complexcomposed of at least one subunit of the signal-transducing glycoproteingp130 and the IL6 receptor (IL6R). IL6 binds to IL6R, which thendimerizes gp130 that triggers the phosphorylation of tyrosine residuesof gp130. At least three major signaling pathways are involved in theformation of the IL6/IL6R/gp130 complex, (1) phosphatidylinositol-3′-kinase (PI3K), (2) mitogen-activated protein kinase (MAK),and (3) Janus tyrosine kinase (JAK)-signal transducer and activator oftranscription 1 and 3 (STAT1 and STAT3) pathway. IL6 is believed to playa role in the development of a wide range of disease or disorders,including but are not limited to, inflammation, autoimmune diseases(e.g. rheumatoid arthritis (RA), Crohn's disease, Castleman's disease,multiple sclerosis, ankylosing spondylitis, psoriatic arthritis andpsoriasis), angiogenesis, cancer (e.g. multiple myeloma, leukemia,breast cancer, pancreatic cancer, lung cancer, ovarian cancer, oralcancer and prostate cancer), tumor metastasis, cancer related cachexia.

As used herein, “rheumatoid arthritis” refers to a type of autoimmunedisease, which is characterized by synovial joint inflammationsthroughout the body. An early symptom of the disease is joint pain,which progresses into joint deformation, or damages in body organs suchas in blood vessels, heart, lungs, skin, and muscles.

As used herein, “angiogenesis” generally refers to the fundamentalprocess by which new blood vessels are formed. Angiogenesis can occur asa normal physiological process during periods of tissue growth, such asan increase in muscle, wound repair and pregnancy, but can also beassociated to a disease condition where the growth of blood vessels isnot beneficial to the health of the patient, such as cancer and diabeticretinopathy.

The term “cancer” as used herein refers to a medical condition mediatedby neoplastic or malignant cell group, proliferation, or metastasis,including solid cancers and non-solid cancers. Examples of cancerinclude but are not limited to, lung cancer, kidney cancer, gastriccancer, breast cancer, brain cancer, prostate cancer, hepatocellularcancer, pancreatic cancer, cervical cancer, ovarian cancer, livercancer, bladder cancer, cancer of the urinary tract, thyroid cancer,melanoma, head and neck cancer, colon cancer, leukemia, lymphomas andmyelomas.

The term “cachexia” refers to a state of general ill health andmalnutrition. It is usually associated with and induced by malignantcancer, and is characterized by severe loss of appetite, dramatic lossof body mass, especially lean body mass, and muscle wasting.

High Affinity Anti-IL6 Antibodies

The present disclosure is based on the identification of a number ofhigh affinity anti-IL6 antibodies, including 1-4-62, Ag1-4-6 (also knownas FB704), and HAg1T-3-10. These anti-IL6 antibodies were found to bindto human IL-6 with high binding affinity (e.g., having a KD value lessthan 10⁻⁸M, preferably less than 10⁻⁹ M) and high specificity, e.g.,binding to other IL6 family cytokines such as those shown in FIG. 3 witha much lower binding affinity as compared with human IL6. Further, theseantibodies were found to significantly reduce IL-6 induced cellproliferation and STATS phosphorylation, angiogenesis and hemoglobinproduction. Moreover, these anti-IL6 antibodies successfully suppressedcancer-induced (e.g., prostate cancer-induced) cachexia, pancreaticcancer growth, and cancer metastasis such as prostate cancer metastasis,significantly enhanced anti-cancer effects of other chemotherapeuticagents such as oxaliplatin, gemcitabine, and docetaxel, and reducedinflammatory cytokine (e.g., MCP-1 and sICAM) and/or VEGF production byHUVEC and PBMC cells and/or synovial fibroblasts, e.g., those obtainedfrom RA patients.

Accordingly, described herein are high affinity antibodies capable ofbinding to (e.g., specifically binding to) human IL6, including 1-4-62,Ag1-4-6, and HAg1T-3-10, and their functional variants. The amino acidsequences of the heavy chain variable region (V_(H)) and light chainvariable region (V_(L)) of each of 1-4-62, Ag1-4-6, and HAg1T-3-10 areshown in Table 1 below. A functional variant of any of these threeantibodies can comprise a V_(H) chain that comprises an amino acidsequence at least 85% (e.g., 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%)identical to that of the V_(H) of 1-4-62, Ag1-4-6, or HAg1T-3-10 (SEQ IDNO:17 or SEQ ID NO:18), a V_(L) chain that has an amino acid sequence atleast 85% (e.g., 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%) identical tothat of the V_(L) of 1-4-62, Ag1-4-6, or HAg1T-3-10 (SEQ ID NO:19 or SEQID NO:20), or both. These variants are capable of binding to human IL6.In some examples, the variants possess similar antigen-binding affinityrelative to the reference antibodies described above (e.g., having aK_(d) less than 1×10⁻⁸, preferably less than 1×10 or 1×10⁻¹⁰ M).

The affinity of the binding is defined by the terms ka (associate rateconstant), kd (dissociation rate constant), or KD (equilibriumdissociation). Typically, specifically binding when used with respect toan antibody refers to an antibody that specifically binds to(“recognizes”) its target(s) with an affinity (KD) value less than 10⁻⁸M, e.g., less than 10⁻⁹ M or 10⁻¹⁰ M. A lower KD value represents ahigher binding affinity (i.e., stronger binding) so that a KD value of10⁻⁹ indicates a higher binding affinity than a KD value of 10⁻⁸.

The “percent identity” of two amino acid sequences is determined usingthe algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad.Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into theNBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol.Biol. 215:403-10, 1990. BLAST protein searches can be performed with theXBLAST program, score=50, wordlength=3 to obtain amino acid sequenceshomologous to the protein molecules of interest. Where gaps existbetween two sequences, Gapped BLAST can be utilized as described inAltschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

Antibodies binding to the same epitopes as 1-4-62, Ag1-4-6, andHAg1T-3-10 are also within the scope of the present disclosure.

In some embodiments, the anti-IL6 antibody comprises a heavy chainvariable region (V_(H)) that comprises a HC CDR3 of MHIDDSNGYXSDAF (SEQID NO:21), in which X is an aromatic amino acid residue such as F, Y, H,or W. In some examples, the HC CDR3 is SEQ ID NO:6 or SEQ ID NO:16. TheV_(H) chain of such an antibody can further comprise a HC CDR1 of SEQ IDNO:2, a HC CDR2 of SEQ ID NO:2, or both.

Alternatively or in addition, the anti-IL6 antibody comprises a lightchain variable region (V_(L)) that comprises a LC CDR3 of SEQ ID NO:13or SEQ ID NO:15. The VL chain of such an antibody can further comprise aLC CDR1 of SEQ ID NO:9, a LC CDR2 of SEQ ID NO:11, or both.

In some embodiments, the anti-IL6 antibodies described herein comprisesthe same heavy chain and light CDRs as antibodies 1-4-62, Ag1-4-6, andHAg1T-3-10 as shown in Table 1 below. In some examples, these antibodiescomprise the same V_(H) and V_(L) chains as 1-4-62, Ag1-4-6, andHAg1T-3-10. The V_(H) and V_(L) chains can be fused with heavy chainC_(H)1 and CL, respectively to form Fab, Fab′ or F(ab′)₂ fragments.Alternatively, the V_(H) and V_(L) chains can be fused with heavy chainconstant region (e.g., human IgG constant chain) and light chainconstant region (a kappa chain) to form full-length antibodies. In otherexamples, the V_(H) and V_(L) chains can be fused, either directly orvia a linker, to form a single chain antibody.

In other embodiments, the functional variants described herein cancontain one or more mutations (e.g., conservative substitutions) in theFRs of the V_(H), the V_(L), or both, as compared to those in antibodies1-4-62, Ag1-4-6, and HAg1T-3-10. Preferably, such mutations do not occurat residues which are predicted to interact with one or more of theCDRs. As known in the art, mutations within the FR regions are unlikelyto affect the antigen-binding activity of the antibody. In someexamples, changes in one or more of the CDR regions of antibodies1-4-62, Ag1-4-6, and HAg1T-3-10 are insubstantial, i.e., substantiallyidentical to a reference sequence.

The term “insubstantial” or “substantially identical” means that therelevant amino acid sequences (e.g., in FRs, CDRs, V_(H), or V_(L)domain) of a variant differ insubstantially (e.g., includingconservative amino acid substitutions) as compared with a referenceantibody such that the variant has substantially similar bindingactivities (e.g., affinity, specificity, or both) and bioactivitiesrelative to the reference antibody. Such a variant may include minoramino acid changes, e.g. 1 or 2 substitutions in a 5 amino acid sequenceof a specified region. Generally, more substitutions can be made in FRregions, in contrast to CDR regions, as long as they do not adverselyimpact the binding function of the antibody (such as reducing thebinding affinity by more than 50% as compared to the original antibody).In some embodiment, the sequence identity can be about 85%, 90%, 95%,96%, 97%, 98%, 99% or higher, between the original and the modifiedantibody. In some embodiments, the modified antibody has the samebinding specificity and has at least 50% of the affinity of the originalantibody. In some examples, the variant includes up to 5 amino acidsubstitutions such as conservative substitutions (e.g., 1, 2, 3, 4, or5) in one or more CDR regions of the V_(H), the V_(L), or both ofantibodies 1-4-62, Ag1-4-6, and HAg1T-3-10.

Conservative substitutions will produce molecules having functional andchemical characteristics similar to those of the molecule from whichsuch modifications are made. For example, a “conservative amino acidsubstitution” may involve a substitution of a native amino acid residuewith another residue such that there is little or no effect on thepolarity or charge of the amino acid residue at that position. Desiredamino acid substitutions (whether conservative or non-conservative) canbe determined by those skilled in the art. For example, amino acidsubstitutions can be used to identify important residues of the moleculesequence, or to increase or decrease the affinity of the moleculesdescribed herein. Variants comprising one or more conservative aminoacid substitutions can be prepared according to methods for alteringpolypeptide sequence known to one of ordinary skill in the art such asare found in references which compile such methods, e.g. MolecularCloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. Conservative substitutions of aminoacids include substitutions made amongst amino acids within thefollowing groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G;(e) S, T; (f) Q, N; and (g) E, D.

The present disclosure also provides antibody variants with improvedbiological properties of the antibody, such as higher binding affinity.Amino acid sequence variants of the antibody can be prepared byintroducing appropriate nucleotide changes into the antibody nucleicacid, or via peptide synthesis. Such modifications include, for example,deletions from, and/or insertions into and/or substitutions of, residueswithin the amino acid sequences of the antibody. Any combination ofdeletion, insertion, and substitution is made to achieve the finalconstruct, provided that the final construct possesses the desiredcharacteristics. Nucleic acid molecules encoding amino acid sequencevariants of the antibody can be prepared by a variety of methods knownin the art. These methods include, but are not limited to,oligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared variant ora non-variant (natural) version of the antibody. In one embodiment, theequilibrium dissociation constant (KD) value of the anti-IL6 antibodiesof the invention is less than 10⁻⁸ M, particularly less than 10⁻⁹ M or10⁻¹⁰ M. The binding affinity may be determined using techniques knownin the art, such as ELISA or biospecific interaction analysis, or othertechniques known in the art.

Exemplary anti-IL6 antibodies as described herein include, but are notlimited to:

(i) an antibody comprising (a) a V_(H) comprising HC CDR1 that comprisesthe amino acid sequence set forth in SEQ ID NO: 2, HC CDR2 thatcomprises the amino acid sequence set forth in SEQ ID NO: 4, and HC CDR3that comprises the amino acid sequence set forth in SEQ ID NO: 6 or SEQID NO: 16; or (b) a V_(L) comprising a LC CDR1 that comprises the aminoacid sequence set forth in SEQ ID NO: 9, a LC CDR2 that comprises theamino acid sequence set forth in SEQ ID NO: 11, and a LC CDR3 thatcomprises the amino acid sequence set forth in SEQ ID NO: 13 or SEQ IDNO: 15;

(ii) an antibody comprising a V_(H) comprising a HC CDR1 that comprisesthe amino acid sequence set forth in SEQ ID NO: 2, a HC CDR2 thatcomprises the amino acid sequence set forth in SEQ ID NO: 4, and a HCCDR3 that comprises the amino acid sequence set forth in SEQ ID NO: 6;

(iii) an antibody comprising a V_(L) comprising a LC CDR1 that comprisesthe amino acid sequence set forth in SEQ ID NO: 9, a LC CDR2 thatcomprises the amino acid sequence set forth in SEQ ID NO: 11, and a LCCDR3 that comprises the amino acid sequence set forth in SEQ ID NO: 13;

(iv) an antibody comprising (a) a V_(H) comprising a HC CDR1 thatcomprises SEQ ID NO: 2, a HC CDR2 that comprises the amino acid sequenceset forth in SEQ ID NO: 4, and a HC CDR3 that comprises the amino acidsequence set forth in SEQ ID NO: 6, and (b) a V_(L) comprising a LC CDR1that comprises the amino acid sequence set forth in SEQ ID NO: 9, a LCCDR2 that comprises the amino acid sequence set forth in SEQ ID NO: 11,and a LC CDR3 that comprises the amino acid sequence set forth in SEQ IDNO: 13;

(v) an antibody comprising a V_(L) comprising a LC CDR1 that comprisesthe amino acid sequence set forth in SEQ ID NO: 9, a LC CDR2 thatcomprises the amino acid sequence set forth in SEQ ID NO: 11, and a LCCDR3 that comprises the amino acid sequence set forth in SEQ ID NO: 15;

(vi) an antibody comprising (a) a V_(H) comprising a HC CDR1 thatcomprises the amino acid sequence set forth in SEQ ID NO: 2, a HC CDR2that comprises the amino acid sequence set forth in SEQ ID NO: 4, and aHC CDR3 that comprises the amino acid sequence set forth in SEQ ID NO:6, and (b) a V_(L) comprising a LC CDR1 that comprises the amino acidsequence set forth in SEQ ID NO: 9, a LC CDR2 that comprises the aminoacid sequence set forth in SEQ ID NO: 11, and a LC CDR3 comprising theamino acid sequence set forth in SEQ ID NO: 15;

(vii) an antibody comprising a V_(H) comprising a HC CDR1 that comprisesthe amino acid sequence set forth in SEQ ID NO: 2, a HC CDR2 thatcomprises the amino acid sequence set forth in SEQ ID NO: 4, and a HCCDR3 that comprises the amino acid sequence set forth in SEQ ID NO: 16;

(viii) an antibody comprising a V_(H) comprising (a) a HC CDR1 thatcomprises the amino acid sequence set forth in SEQ ID NO: 2, a HC CDR2that comprises the amino acid sequence set forth in SEQ ID NO: 4, and aHC CDR3 that comprises the amino acid sequence set forth in SEQ ID NO:16, and (b) a V_(L) comprising a LC CDR1 that comprises the amino acidsequence set forth in SEQ ID NO: 9, a LC CDR2 that comprises the aminoacid sequence set forth in SEQ ID NO: 11, and a LC CDR3 that comprisesthe amino acid sequence set forth in SEQ ID NO: 15.

Any of the anti-IL6 antibodies described herein can be examined todetermine their properties, such as antigen-binding activity,antigen-binding specificity, and biological functions, following routinemethods, e.g., those described in the examples below.

Any of the anti-IL6 antibodies described herein can be modified tocontain additional nonproteinaceous moieties that are known in the artand readily available, e.g., by PEGylation, hyperglycosylation, and thelike. Modifications that can enhance serum half-life are of interest.

Also disclosed herein are nucleic acids encoding any of the anti-IL6antibodies described herein, vectors such as expression vectorscomprising these nucleic acids, and host cells comprising the vectors.In one example, both the heavy and light chain coding sequences (e.g.,sequences encoding a V_(H) and a V_(L), a V_(H)—C_(H)1 and aV_(L)-C_(L), or a full-length heavy chain and a full-length light chain)are included in one expression vector. In another example, each of theheavy and light chains of the antibody is cloned into an individualvector. In the latter case, the expression vectors encoding the heavyand light chains can be co-transfected into one host cell for expressionof both chains, which can be assembled to form intact antibodies eitherin vivo or in vitro. Alternatively, the expression vector encoding theheavy chain and that encoding the light chain can be introduced intodifferent host cells for expression each of the heavy and light chains,which can then be purified and assembled to form intact antibodies invitro.

Numerous methods known to those skilled in the art are available forobtaining antibodies or antigen-binding fragments thereof. For example,antibodies can be produced using recombinant DNA methods. Monoclonalantibodies may also be produced by generation of hybridomas. Hybridomasformed in this manner are then screened using standard methods, such asenzyme-linked immunosorbent assay (ELISA) to identify one or morehybridomas that produce an antibody that specifically binds with aspecified antigen. In addition, phage display systems can be used toscreen for single chain antibodies.

Alternatively, any of the anti-IL6 antibodies can be prepared viaconventional methodology, e.g., recombination technology. For example,the polypeptide sequences provided herein (see, e.g., Table 1) for theexemplary antibodies described herein can be used to obtain suitablenucleic acid sequences encoding such, and the nucleic acids sequencescan be cloned into suitable expression vectors via conventionalrecombinant technology for producing the antibodies in suitable hostcells (e.g., bacterial cells, yeast cells, or mammalian cells such asCHO cells) by routine methods. The antibodies thus prepared can beisolated from the cells or the culture supernatants and their bindingfeatures and bioactivities can be examined also by routine technology.

In one example, phage display systems are used to select IL6 singlechain antibodies. Once isolated, polynucleotides encoding specific IL6scFvs may be cloned into expression vectors designed to express fulllength immunoglobulins and fragments thereof having the desiredspecificity. Briefly, the V_(H) and V_(L) polynucleotides of the singlechain antibody are cloned into an immunoglobulin scaffold (i.e., IgG)vector, expressed, and dimerized so as to “convert” the single chaininto a full antibody. The immunoglobulin scaffold may be selected fromany of the five major classes of immunoglobulins (IgA, IgD, IgE, IgG andIgM) as needed. Methods for the conversion of scFvs into intactimmunoglobulin molecules are well known, for example, as described in,WO 94/11523, WO 97/9351 or EP 0481790.

The recombinant vectors for expression the antibodies described hereintypically contain a nucleic acid encoding the antibody amino acidsequences operably linked to a promoter, either constitutive orinducible. The vectors can be suitable for replication and integrationin prokaryotes, eukaryotes, or both. Typical vectors containtranscription and translation terminators, initiation sequences, andpromoters useful for regulation of the expression of the nucleic acidencoding the antibody. The vectors optionally contain generic expressioncassettes containing at least one independent terminator sequence,sequences permitting replication of the cassette in both eukaryotes andprokaryotes, i.e., shuttle vectors, and selection markers for bothprokaryotic and eukaryotic systems.

Recombinant anti-IL6 antibodies as described herein may be produced inprokaryotic or eukaryotic expression systems, such as bacteria, yeast,filamentous fungi, insect, and mammalian cells. It is not necessary thatthe recombinant antibodies of the invention be glycosylated or expressedin eukaryotic cells; however, expression in mammalian cells is generallypreferred. Examples of useful mammalian host cell lines are humanembryonic kidney line (293 cells), baby hamster kidney cells (BHKcells), Chinese hamster ovary cells/− or +DHFR (CHO, CHO-S, CHO-DG44,Flp-in CHO cells), African green monkey kidney cells (VERO cells), andhuman liver cells (Hep G2 cells). Host cells are transformed ortransfected with the vectors (for example, by chemical transfection orelectroporation methods) and cultured in conventional nutrient media (ormodified as appropriate) for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.The antibody protein as produced can be further isolated or purified toobtain preparations that substantially homogeneous for further assaysand applications. Standard protein purification methods known in the artcan be used. For example, suitable purification procedures may includefractionation on immunoaffinity or ion-exchange columns, ethanolprecipitation, high-performance liquid chromatography (HPLC), sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), ammoniumsulfate precipitation, and gel filtration.

When a full-length antibody is desired, coding sequences of any of theanti-IL6 V_(H) and V_(L) chains described herein can be linked to thecoding sequences of the Fc region of a human immunoglobulin and theresultant gene encoding a full-length antibody heavy and light chainscan be expressed and assembled in a suitable host cell, e.g., a plantcell, a mammalian cell, a yeast cell, or an insect cell.

Antigen-binding fragments can be prepared via routine methods. Forexample, F(ab′)₂ fragments can be produced by pepsin digestion of anfull-length antibody molecule, and Fab fragments that can be generatedby reducing the disulfide bridges of F(ab′)₂ fragments. Alternatively,such fragments can be prepared via recombinant technology by expressingthe heavy and light chain fragments in suitable host cells (e.g., E.coli, yeast, mammalian, plant, or insect cells) and have them assembledto form the desired antigen-binding fragments either in vivo or invitro.

A single-chain antibody can be prepared via recombinant technology bylinking a nucleotide sequence coding for a heavy chain variable regionand a nucleotide sequence coding for a light chain variable region.Preferably, a flexible linker is incorporated between the two variableregions.

Uses of Anti-IL6 Antibodies

Anti-IL6 antibodies as described herein were found to bind IL6 and actas antagonists to IL6 and regulate IL6 dependent signaling pathway. Inparticularly, those antibodies were found to significantly inhibitIL6-dependent cell proliferation (e.g., pancreatic cancer cell growth)and phosphorylated STAT signaling transduction, decrease IL6-inducedangiogenic formation in vivo, and suppress human tumor metastasis (e.g.,prostate cancer metastasis). Further, these antibodies exhibitedsynergistic efficacy when co-used with a chemotherapeutic agent, such asoxaliplatin, gemticibine, and docetaxel, in animal models of humanpancreatic tumor.

Therefore, the anti-IL6 antibodies described herein can be used intreating a disease or condition associated with IL6, including, but arenot limited to, inflammatory disorder, autoimmune diseases (e.g.rheumatoid arthritis (RA), Crohn's disease, Castleman's disease,multiple sclerosis, ankylosing spondylitis, psoriatic arthritis andpsoriasis), angiogenesis, cancer such as a solid tumor (e.g. multiplemyeloma, leukemia, breast cancer, pancreatic cancer, lung cancer,ovarian cancer, oral cancer and prostate cancer), tumor metastasis, andcancer related cachexia.

To practice the treatment methods described herein, any of the anti-IL6antibodies (e.g., an antibody having the same CDRs or same V_(H) andV_(L) chains as antibodies 1-4-62, Ag1-4-6, or HAg1T-3-10), or a nucleicacid(s) (e.g., an expression vector) encoding such an antibody can beformulated into a pharmaceutical composition with one or morepharmaceutically acceptable carriers. “Pharmaceutically acceptable” asused herein means that the carrier is compatible with the activeingredient contained in the composition, preferably capable ofstabilizing the active ingredient, and not deleterious to the subject tobe treated. The carrier may serve as a diluent, vehicle, excipient, ormedium for the active ingredient. Some examples of suitable carriersinclude physiologically compatible buffers, such as Hank's solution,Ringer's solution, physiological saline buffer, lactose, dextrose,sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,alginates, tragacanth, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, andmethyl cellulose. The pharmaceutical composition can additionallyinclude lubricating agents such as talc, magnesium stearate, and mineraloil; wetting agents; emulsifying and suspending agents; preservingagents such as methyl- and propylhydroxy-benzoates; sweetening agents;and flavoring agents. See, e.g., Remington's Pharmaceutical Sciences,Edition 16, Mack Publishing Co., Easton, Pa. (1980); and Goodman andGilman's “The Pharmacological Basis of Therapeutics”, Tenth Edition,Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173,2001.

The pharmaceutical composition according to the invention can be in theform of tablets, pills, powders, lozenges, sachets, cachets, elixirs,suspensions, emulsions, solutions, syrups, soft and hard gelatincapsules, suppositories, sterile injectable solutions, and packagedpowders. The pharmaceutical composition of the invention may bedelivered through any physiologically acceptable route. These routes caninclude, but are by no means limited to parenteral administration,systemic administration, oral administration, nasal administration,rectal administration, intraperitoneal injection, intravascularinjection, subcutaneous injection, transcutaneous administration,inhalation administration, and intramuscular injection. The term“parenteral” as used herein includes subcutaneous, intracutaneous,intravenous, intramuscular, intraarticular, intraarterial,intrasynovial, intrasternal, intrathecal, intralesional, andintracranial injection or infusion techniques.

The pharmaceutical compositions, formulated for therapeutic uses, may beprepared for storage by mixing an agent having the desired degree ofpurity with optional pharmaceutically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients, or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In some embodiments, the method described herein aims at treatingcancer, such as prostate cancer. A human subject who needs thistreatment can be a patient suffering from or is suspected of havingcancer. In some examples, the amount of the anti-IL6 antibody describedherein is effective in inhibiting IL6-induced cell proliferation by atleast 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% as compared to ablank control. In other embodiments, the amount of the anti-IL6 antibodydescribed herein is effective in inhibiting STAT3 phosphorylation by atleast 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500%. In some examples,the amount of the anti-IL6 antibody described herein is effective ininhibiting IL6-induced angiogenesis, cancer-induced cachexia (e.g.,prostate cancer-induced cachexia), cancer metastasis (prostate cancermetastasis), or a combination thereof.

In other embodiment, the method described herein is for treating anautoimmune disease, such as rheumatoid arthritis. In another example,the subject is a human rheumatoid arthritis patient who suppers from oris suspected of having the disease. In some example, the amount of theanti-IL6 antibody described herein is sufficient in reducing theproduction of inflammatory cytokines such as MCP-1 and/or sICAM, e.g.,by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500%.

To treating a target disease such as cancer or rheumatoid arthritis, aneffective amount of the pharmaceutical composition noted above can beadministered to a subject (e.g., a human) in need of the treatment via asuitable route. A human subject who needs the treatment may be a humanpatient having, at risk for, or suspected of having a disorderassociated with IL6. Such a patient can be identified by routine medicalexamination.

Any of the anti-IL6 antibodies as described herein may be used incombination with another therapeutic agent. The term “in combination” inthis context means that the antibody composition and the therapeuticagent are given either simultaneously or sequentially. For example, thecombination therapy can include at least one anti-IL6 antibodyco-formulated with and/or co-administered with, at least one additionaltherapeutic agent. In one embodiment, the additional agent is a cancerchemotherapeutic agent e.g. oxaliplatin, gemcitabine, docetaxel. Inanother embodiment, the additional agent can be disease modifyingantirheumatic drugs (DMARDs) e.g. methotrexate, azathioprine,chloroquine, hydroxychloroquine, cyclosporin A, sulfasalazine, for RAtreatment. Such combination therapies may advantageously utilize lowerdosages of the administered therapeutic agents, thus preventing possibletoxicities or complications associated with the various monotherapies.Moreover, the additional therapeutic agents disclosed herein may act onpathways in addition to or distinct from the IL6/I6R/gp130 pathway, andthus are expected to enhance and/or synergize with the effects of theanti-IL6 antibodies.

When the antibody composition described here is co-used with a secondtherapeutic agent, a sub-therapeutic dosage of either the composition orof the second agent, or a sub-therapeutic dosage of both, can be used inthe treatment of a subject having, or at risk of developing a disease ordisorder associated with the cell signaling mediated by IL6. A“sub-therapeutic dose” as used herein refers to a dosage, which is lessthan that dosage which would produce a therapeutic result in the subjectif administered in the absence of the other agent or agents. Thus, thesub-therapeutic dose of an agent is one which would not produce thedesired therapeutic result in the subject in the absence of theadministration of the anti-IL6 antibody described herein. Therapeuticdoses of many agents that are in clinical use are well known in thefield of medicine, and additional therapeutic doses can be determined bythose of skill without undue experimentation. Therapeutic dosages havebeen extensively described in references such as Remington'sPharmaceutical Sciences, 18th ed., 1990; as well as many other medicalreferences relied upon by the medical profession as guidance for thetreatment of diseases and disorders.

Conventional methods, known to those of ordinary skill in the art ofmedicine, can be used to administer the pharmaceutical composition tothe subject, depending upon the type of diseases to be treated or thesite of the disease.

Injectable compositions may contain various carriers such as vegetableoils, dimethylactamide, dimethyformamide, ethyl lactate, ethylcarbonate, isopropyl myristate, ethanol, and polyols (glycerol,propylene glycol, liquid polyethylene glycol, and the like). Forintravenous injection, water soluble antibodies can be administered bythe drip method, whereby a pharmaceutical formulation containing theantibody and a physiologically acceptable excipients is infused.Physiologically acceptable excipients may include, for example, 5%dextrose, 0.9% saline, Ringer's solution or other suitable excipients.Intramuscular preparations, e.g., a sterile formulation of a suitablesoluble salt form of the antibody, can be dissolved and administered ina pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or5% glucose solution.

When a nucleic acid(s) encoding an anti-IL6 antibody as described hereinis used as the therapeutic agent, the nucleic acid(s) or a vector(s)expressing the antibody can be delivered to a subject by methods, suchas that described in Akhtar et al., 1992, Trends Cell Bio. 2, 139. Forexample, it can be introduced into cells using liposomes, hydrogels,cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres.Alternatively, the nucleic acid or vector can be locally delivered bydirect injection or by use of an infusion pump. Other approaches includeemploying various transport and carrier systems, for example through theuse of conjugates and biodegradable polymers.

To facilitate delivery, any of the anti-IL6 antibody or its encodingnucleic acids can be conjugated with a chaperon agent. As used herein,“conjugated” means two entities are associated, preferably withsufficient affinity that the therapeutic benefit of the associationbetween the two entities is realized. Conjugated includes covalent ornoncovalent bonding as well as other forms of association, such asentrapment of one entity on or within the other, or of either or bothentities on or within a third entity (e.g., a micelle).

The chaperon agent can be a naturally occurring substance, such as aprotein (e.g., human serum albumin, low-density lipoprotein, orglobulin), carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin or hyaluronic acid), or lipid. It can also be arecombinant or synthetic molecule, such as a synthetic polymer, e.g., asynthetic polyamino acid. Examples of polyamino acids include polylysine(PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acidanhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinylether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamidecopolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA),polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamidepolymers, and polyphosphazine.

In one example, the chaperon agent is a micelle, liposome, nanoparticle,or microsphere, in which the oligonucleotide/interfering RNA isencapsulated. Methods for preparing such a micelle, liposome,nanoparticle, or microsphere are well known in the art. See, e.g., U.S.Pat. Nos. 5,108,921; 5,354,844; 5,416,016; and 5,527,5285.

In another example, the chaperon agent serves as a substrate forattachment of one or more of a fusogenic or condensing agent.

A fusogenic agent is responsive to the local pH. For instance, uponencountering the pH within an endosome, it can cause a physical changein its immediate environment, e.g., a change in osmotic properties whichdisrupts or increases the permeability of the endosome membrane, therebyfacilitating release of the antisense oligonucleotide into host cell'scytoplasm. A preferred fusogenic agent changes charge, e.g., becomesprotonated at a pH lower than a physiological range (e.g., at pH4.5-6.5). Fusogenic agents can be molecules containing an amino groupcapable of undergoing a change of charge (e.g., protonation) whenexposed to a specific pH range. Such fusogenic agents include polymershaving polyamino chains (e.g., polyethyleneimine) and membranedisruptive agents (e.g., mellittin). Other examples includepolyhistidine, polyimidazole, polypyridine, polypropyleneimine, and apolyacetal substance (e.g., a cationic polyacetal).

A condensing agent interacts with the antisense oligonucleotide, causingit to condense (e.g., reduce the size of the oligonucleotide), thusprotecting it against degradation. Preferably, the condensing agentincludes a moiety (e.g., a charged moiety) that interacts with theoligonucleotide via, e.g., ionic interactions. Examples of condensingagents include polylysine, spermine, spermidine, polyamine orquarternary salt thereof, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationiclipid, cationic porphyrin, and alpha helical peptide.

The anti-IL6 antibodies described herein may also be used to detect thepresence of IL6 in biological samples. Antibody-based detection methodsare well known in the art, and include, for example, ELISA, immunoblots,radioimmunoassays, mmunofluorescence, immunoprecipitation, and otherrelated techniques. The antibodies may be provided in a diagnostic kitthat incorporates at least one other components to detect the protein.The kit may also contain packaging, instructions, or other material toaid the detection of the protein and use of the kit.

Antibodies may be modified with detectable markers, including ligandgroups (e.g., biotin), radioisotopes, fluorophores, or enzymes. Enzymesare detected by their activity. For example, horseradish peroxidase isdetected by its ability to convert the substrate, tetramethylbenzidine(TMB), to a blue pigment, which is quantifiable with aspectrophotometer. Antibodies can also be functionally linked (e.g., bygenetic fusion, chemical coupling, noncovalent association or otherwise)to at least one other molecule(s), such as another antibody (e.g., abispecific or a multispecific antibody), cytotoxic or cytostatic agents,toxins, radioisotopes and the like.

Kits

The present disclosure also provides kits for use in treating diseasesassociated with IL6. Such kits can include one or more containerscomprising an anti-IL6 antibody (e.g., 1-4-62, Ag1-4-6, and HAg1T-3-10)or its encoding nucleic acid.

In some embodiments, the kit can comprise instructions for use inaccordance with any of the methods described herein. The includedinstructions can comprise a description of administration of theanti-IL6 antibody to treat, delay the onset, or alleviate a diseaseassociated with IL6 such as cancer (e.g., prostate cancer) or anautoimmune disease (e.g., RA) according to any of the methods describedherein. The kit may further comprise a description of selecting anindividual suitable for treatment based on identifying whether thatindividual has the disease. In still other embodiments, the instructionscomprise a description of administering an anti-IL6 antibody anindividual at risk of the disease.

The instructions relating to the use of an anti-IL6 antibody generallyinclude information as to dosage, dosing schedule, and route ofadministration for the intended treatment. The containers may be unitdoses, bulk packages (e.g., multi-dose packages) or sub-unit doses.Instructions supplied in the kits of the invention are typically writteninstructions on a label or package insert (e.g., a paper sheet includedin the kit), but machine-readable instructions (e.g., instructionscarried on a magnetic or optical storage disk) are also acceptable.

The label or package insert indicates that the composition is used fortreating, delaying the onset and/or alleviating liver fibrosis orcirrhosis. Instructions may be provided for practicing any of themethods described herein.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Alsocontemplated are packages for use in combination with a specific device,such as an inhaler, nasal administration device (e.g., an atomizer) oran infusion device such as a minipump. A kit may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Thecontainer may also have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an anti-IL6 antibody.

Any of the kit described herein may further include an additionaltherapeutic agent, such as an anti-cancer drug (e.g., oxaliplatin,gemcitabine, or docetaxel) or a DMARD (e.g., methotrexate, azathioprine,chloroquine, hydroxychloroquine, cyclosporine A, or sulfasalazine).

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container. In someembodiments, the invention provides articles of manufacture comprisingcontents of the kits described above.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

Example 1: Identification of High Affinity Antibodies Binding to HumanIL6

A phage-displayed human naïve scFv library was constructed as followsfor identifying high affinity human antibodies capable of binding tohuman IL6.

mRNAs were isolated from peripheral blood lymphocytes of 151 healthdonors and cDNAs were synthesized from such by M-MuLV reversetranscriptase (Fermentas) using oligo dT primers. V_(H) and V_(L) geneswere amplified, assembled, and ligated into a phagemid vector bystandard protocols with some modifications. The ligated products wereintroduced into TG1 E-coli cells via electroporation. Afterwards, the E.coli cells were recovered and incubated in the 2YT medium containing 100mg/ml ampicillin and 2% glucose. M13KO7 helper phage particles wereadded to the culture to generate scFv-phage particles, thus producing anscFv library. The diversity of the library was determined by sequencingover 1,000 clones.

The scFv library was subjected to four rounds of biopanning as follows.Wells were coated with recombinant IL6 in 0.1M NaHCO3 buffer at 4° C.overnight and then rinsed twice with 300 μl of phosphate buffered saline(PBS). The wells were blocked with 1% bovine serum albumin (BSA) in PBSfor 1 h at 37° C.; added with 100 μl of the phage particles (2×10¹¹ pfu)in PBS containing 1% BSA, and rocked for 1 h at room temperature (RT).The wells were then washed 6 times with 300 μl 0.5% (w/v) Tween 20/PBS.Bound phages were eluted and amplified by infection with E coli TG1. Theinfected cells were rescued by M13KO7. The resultant phage particleswere concentrated via PEG-precipitation and used for the next round ofbiopanning.

In the fourth and fifth rounds of biopanning, the phage clones wereexamined by Enzyme-linked immunosorbent assay (ELISA) screening fortheir antigen-binding specificities. ELISA plates were coated withrecombinant human IL6 or BSA at 2 μg/ml in 0.1M NaHCO3 buffer at 4° C.overnight. After being washed by PBS, the wells were blocked with 1%(w/v) BSA in PBS for 1 h at RT. Phage clones and scFv fragments wereadded to the plates, which were incubated for 1 h at RT. The plate waswashed by PBS containing 0.1% (v/v) Tween 20 and then probed withHRP-labeled goat anti-human IgG (KPL) diluted 1:10,000 in 1% BSA-PBS for40 min at RT. The plate was again washed and3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution was added todevelop color. Stop solution (1M H₂SO₄) was then added and theabsorption at 450 nm was quantitated using an automated plate photometer(Bio-Rad).

After four rounds of biopanning, more than eight hundred phage cloneswere identified as capable of binding to the rhIL6 by the ELISA assaydescribed above. 29 phage clones were selected for sequence analysis todetermine the V_(H) and V_(L) sequences encoding anti-IL6 antibodies. Anumber of unique clones were identified; their binding activity andspecificity were determined by a comparative ELISA assay.

A parent phage clone identified from the biopanning procedures describedabove, clone 1-4-62, was selected for affinity maturation to producehigh affinity anti-IL6 antibodies via construction via site-directedmutagenesis of randomized V_(L)/V_(H) CDR phage antibody libraries, eachcontaining more than 0.9×10⁹ variant antibodies. The libraries wereconstructed using V_(L) and V_(H) forward primers containing randomizedsequences at the CDR regions based on the V_(L) and V_(H) sequences ofclone 1-4-62, following standard protocols below. The CDR randomizedlibraries were subjected to the biopanning procedures under highstringency conditions as described herein and at least two high affinityclones, Ag1-4-6 (FB704) and Hag1T-3-10 were identified as having higherbinding activities relative to the parent clone.

The complementary determining regions 1-3 (CDR 1-3) and frameworkregions 1-4 (FW1-4) for both the V_(H) and V_(L) domains for the threeanti-IL6 antibodies, 1-4-62, Ag1-4-6, and Hag1T-3-10, are provided inTable 1 below:

TABLE 1Amino acid sequences of V_(H) and V_(L) domains for anti-IL6 antibodiesV_(H) domain FWI CDR1 FW2 CDR2 1-4-62 EVQLVESGPALVKPTQT TGGMSVS (SEQ IDWIRQPPGKALEWL RIDWDDDKFYTPS LTLTCTFSGFSLS (SEQ NO: 2) A (SEQ ID NO: 3)LKT (SEQ ID NO: ID NO: 1) 4) Ag1-4-6 EVQLVESGPALVKPTQT TGGMSVS (SEQ IDWIRQPPGKALEWL RIDWDDDKFYTPS LTLTCTFSGFSLS (SEQ NO: 2) A (SEQ ID NO: 3)LKT (SEQ ID NO: ID NO: 1) 4) HAg1T-3-10 EVQLVESGPALVKPTQTTGGMSVS (SEQ ID WIRQPPGKALEWL RIDWDDDKFYTPS LTLTCTFSGFSLS (SEQ NO: 2)A (SEQ ID NO: 3) LKT (SEQ ID NO: ID NO: 1) 4) FW3 CDR3 FW4 1-4-62RLTISRDTSKNQVVLIMT MHIDDSNGYYSDAF WGQGTMVTVSS NMDPVDTATYYCARHI (SEG ID NO: 6) (SEQ ID NO: 7) (SEQ ID NO: 5) Ag1-4-6RLTISRDTSKNQVVLIMT MHIDDSNGYYSDAF WGQGTMVTVSS NMDPVDTATYYCARHI (SEG ID NO: 6) (SEQ ID NO: 7) (SEQ ID NO: 5) HAg1T-3-10RLTISRDTSKNQVVLIMT MHIDDSNGYFSDAF WGQGTMVTVSS NMDPVDTATYYCARHI (SEG ID NO: 16) (SEQ ID NO: 7) (SEQ ID NO: 5) V_(L) domain FWI CDR1FW2 CDR2 1-4-62 EIVLTQSPATLSVSPGER RDSGSVSSTSLA WYQQKSGQAPRLLDTSNRAT (SEQ ID VTLSC (SEQ ID NO: 8) (SEQ ID NO: 9) IY (SEQ ID NO: 10)NO: 11) Ag1-4-6 EIVLTQSPATLSVSPGER RDSGSVSSTSLA WYQQKSGQAPRLLDTSNRAT (SEQ ID VTLSC (SEQ ID NO: 8) (SEQ ID NO: 9) IY (SEQ ID NO: 10)NO: 11) HAg1T-3-10 EIVLTQSPATLSVSPGER RDSGSVSSTSLA WYQQKSGQAPRLLDTSNRAT (SEQ ID VTLSC (SEQ ID NO: 8) (SEQ ID NO: 9) IY (SEQ ID NO: 10)NO: 11) FW3 CDR3 FW4 1-4-62 GIPARFSGGGSGTDFTL LVRNNWPPRFT FGQGTKVEIKTISSLEPEDFAVYYC (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO: 12) Ag1-4-6GIPARFSGGGSGTDFTL SFVSRPYPRFT (SEQ FGQGTKVEIK TISSLEPEDFAVYYC ID NO: 15)(SEQ ID NO: 14) (SEQ ID NO: 12) HAg1T-3-10 GIPARFSGGGSGTDFTLSFVSRPYPRFT (SEQ FGQGTKVEIK TISSLEPEDFAVYYC ID NO: 15) (SEQ ID NO: 14)(SEQ ID NO: 12)

Example 2: Characterization of High Affinity Antibodies Binding to HumanIL6 Materials and Methods (i) Cell Lines and Antibodies

The human myeloma cells, U266 cell line (BCRC 60437), were obtained fromthe Bioresource Collection and Research Center (BCRC) in Taiwan and werecultured in RPMI 1640 (Biowet) supplemented with 10% fetal bovine serum.

The IL6-dependent B cell hybridoma, B9, was cultured in RPMI 1640supplemented with 5% fetal bovine serum plus 50 pg/ml recombinant humanIL6.

Human umbilical vein endothelial cells (HUVECs) were purchased fromLonza and were grown on gelatin-coated petridish in EGM™-2 singleQuots®medium (Lonza).

The IgG producing cells, FreeStyle™ CHO cell line (Invitrogen), werecultured in FreeStyle™ CHO expression medium (Invitrogen) with 8 mML-glutamine.

Flp-in CHO cell line (Invitrogen) were cultured in Ham's F12(Invitrogen) with 10% fetal bovine serum.

A control anti-IL6 antibody, BE8, was purchased from Diaclone. Actemra,an antibody drug (anti-IL6 antibodies) approved by the U.S. Food andDrug Administration (FDA) for treating rheumatoid arthritis, waspurchased from Universitatsklinikum Heidelberg, Germany. Human IgG1nucleotide was purchased from Sigma.

(ii) Production of Fully Human Anti-IL6 IgG1 antibodies Four expressedvectors (pA01-Kappa, pAO2-Gamma, p2CMV intermediate and modifiedpcDNAFRT) were constructed for producing human IgG in mammalian cells.The whole light chain gene (containing a Kappa constant region) and thewhole heavy chain gene (containing a gamma constant region) encodinganti-IL6 candidate antibodies were cloned from pcANTAB5E into pA01-Kappaand pAO2-Gamma separately for transient antibody expression. Theseexpression vectors were introduced into FreeStyle™ CHO-S cells byGenJet™ Plus reagent (SignalGen).

The whole light chain and heavy chain genes were first cloned into p2CMVintermediate vector and then cloned into a modified pcDNAFRT singleexpression vector for stable antibody expression. The pcDNAFRT vectorwas transfected into CHO cells by GenJet™ Plus reagent and positiveclones were selected with Hygromycin B (Invitrogen) for a longer periodof time expression. The culture supernatant was harvested and purifiedby MabSelect SuRe and protein A columns (GE Healthcare).

(iii) Binding and Competition Analysis

ELISA and Western blot were performed to examine the binding activity ofanti-IL6 human IgG antibodies. Briefly, plates were coated withrecombinant human IL6 (R&D Systems) at 2 μg/ml. After being washed byPBS, the wells were blocked with 1% (w/v) BSA in PBS for 1 h at RT.Purified anti-IL6 antibodies in serial dilutions (starting concentrationof 2 μg/ml) were placed into the wells and incubated at RT for 1 h. Theplate was washed by PBS containing 0.1% (w/v) Tween 20 and then probedwith HRP-labeled goat anti-human IgG (KPL), which was diluted 1:10,000in 1% BSA-PBS, for 40 min at RT. The plate was again washed and a TMBsubstrate solution was added to develop color. Stop solution (1M H2SO4)was then added and the absorption at 450 nm was quantitated using anautomated plate photometer (Bio-Rad).

Recombinant IL6 protein was boiled in 5× reducing sample buffer (0.15 MTris-HCl, pH 6.8, 50% glycerol, 10% SDS, 0.71 M 2-mercaptoethanol,0.095% bromophenol blue) for 10 min, and subjected to SDS-PAGE(Bio-Rad). The proteins were then transferred to a nitrocellulosemembrane (Bio-Rad), and immunoblotted with the fully-human anti-IL6antibodies described herein. After being incubated with HRP-labeled goatanti-rabbit (Thermo) or HRP-labeled goat anti-mouse (KPL) antibody, themembranes were developed by enhanced chemiluminescence (GE Healthcare).

Plates were coated with recombinant human IL6 receptor-α (R&D Systems)at 2 μg/ml in 0.1M NaHCO3 buffer at 4° C. overnight. After being washedby PBS, the wells were blocked with 1% (w/v) BSA in PBS for 1 h at RT.Purified anti-IL6 antibodies in two-fold serial dilutions at a startingconcentration of 2 μg/ml were pre-incubated with rhIL6 (0.5 μg/ml) at RTfor 1 hr. The mixtures were the placed into the plate and incubated atRT for 1 h. The plate was washed by PBS containing 0.1% (v/v) Tween 20and then probed with mouse anti-human IL-6 IgG2a (Abcam) diluted 1:2,000in 1% BSA-PBS for 1 h at RT. The plate was washed and then probed withHRP-labeled goat anti-mouse IgG (Thermo) diluted 1:10,000 in 1% BSA-PBSfor 40 min at RT. The plate was again washed and a TMB substratesolution was added to develop color. Stop solution (1M H₂SO₄) was thenadded and the absorption at 450 nm quantitated using an automated platephotometer.

(iv) Cell Signaling Transduction Assay

U266 cells (1×106/ml) were washed twice with serum-free RPMI 1640 andcultured for 2 h in the absence of fetal bovine serum and growthfactors. Cells were stimulated with rhIL6 (5 ng/ml) for 30 min at 37° C.and then treated with or without anti-IL6 antibodies. Cells were washedwith ice-cold PBS, and then lysed in RIPA buffer (Thermo) supplementedwith protease and phosphatase inhibitor (Roche). Western blot analysiswas performed using anti-pSTAT3, and anti-STAT3 (Cell SignalingTechnology) antibodies. After incubating with HRP-labeled goatanti-rabbit (Thermo) or HRP-labeled goat anti-mouse antibody (Thermo),the membranes were developed by enhanced chemiluminescence (GEHealthcare).

(v) Cell Proliferation Assay

Murine B9 cells (5×104/ml) were seed in 96-well plates and incubatedwith rhIL6 (10 pg/ml). The IL6 receptor of murine B9 cell could bestimulated by human IL6. Anti-IL6 antibodies at various concentrationsor control IgG antibodies were placed into to the wells. The cells werethen cultured at 37° C. for 72 h. The cell viability was detected byWater Soluble Tetrazolium (WST-1) assay (Roche) according tomanufacturer's instruction. All experiments were carried out intriplicates.

(vi) Biacore Analysis

IgG binding affinity was measured by using human antibody capture kit(GE healthcare) in BIAcore T200 system (GE healthcare). The mouseanti-human IgG was immobilized on a CMS sensor chip via amine coupling.Approximately 700 RU of purified anti-IL6 IgG in HBS-EP+ buffer werecaptured onto the immobilized surface. The recombinant human IL6 (R&DSystems) at concentrations ranging from 25.6 nM to 0.4 nM in HBS-EP+buffer was injected for 2 minutes using a flow rate of 30 μl/min.Dissociation of bound antigen in HBS⁻EP⁺ buffer flow was followed for 7minutes. The surfaces IgG were regenerated after each cycle usingregeneration solution (3M MgCl₂). The dissociation constant (KD) wascalculated as kd/ka.

(vii) Specificity Analysis

Plates were coated with recombinant human IL3, IL4, IL5, IL6, IL11,IL17A, CNTF, OSM, IGF-1 (R&D Systems), IL2, FGF (Prospec), VEGF, TNF-α,EGF (Peprotech) at 2 μg/ml in 0.1M NaHCO₃ buffer at 4° C. overnight.After being washed by PBS, the wells were blocked with 1% (w/v) BSA inPBS at RT for 1 h. Purified anti-IL6 antibodies in four-fold serialdilutions at a starting concentration of 2 μg/ml were placed into thewells and incubated at RT for 1 h. The plate was washed in PBScontaining 0.1% (w/v) Tween 20 and then probed with HRP-labeled goatanti-human IgG (KPL) diluted 1:10,000 in 1% BSA-PBS for 40 min at RT.The plate was again washed and a TMB substrate solution was added todevelop color. Stop solution (1M H₂SO₄) was then added and theabsorption at 450 nm quantitated using VERSA max (Molecular Devices).

Results (a) Binding Activity of Anti-IL6 IgG Antibodies

To explore the binding specificity of anti-IL6 antibodies, V_(H) andV_(L) genes encoding the selected scFv clones were fused with kappa andgamma constant region genes to produce whole human IgG1 antibodies asdescribed herein. ELISA and western blot assays showed that thoseanti-IL6 antibodies could bind human recombinant IL6 protein andexhibited neutralization activity in a dose-dependent manner.

The BIAcore analysis showed the average of association rate constants(ka) of clones Ag1-4-6 and Hag1T-3-10 were enhanced from 4.38×10⁻⁵ to2.24×10⁻⁶ M⁻¹s⁻¹ as compared with the parent 1-4-62 clone. Thedissociation rate constants (kd) were enhanced from 1.66×10⁻³ to 6×10⁻⁴s⁻¹. The equilibrium dissociation constants (KD) were improved from 3.85to 0.27 nM. See Table 2 below.

TABLE 2 Associate rate constants (ka) and dissociation constants (kd) ofanti-IL6 antibodies Antibody Ka (1/Ms) Kd (1/s) KD (nM) 1-4-62 4.38E+051.66E−03 3.85 Ag1-4-6 1.61E+06 1.21E−03 0.75 HAg1T-3-10 2.24E+066.00E−04 0.27

(b) Binding Specificity of Anti-IL6 Antibodies

An ELISA assay as described herein was performed to confirm the bindingspecificity of high-affinity anti-IL6 antibodies described herein.Briefly, plates were coated with different cytokines as indicated inFIG. 3A (IL6 family cytokines) and FIG. 3B (non-IL6 family cytokines)and the binding activities of the FB704 antibody clone (i.e. Ag1-4-6) tothose cytokines were examined. FB704 showed high binding activity tohuman IL6 protein but not to other cytokines (FIGS. 3A and 3B),indicating its binding specificity to human IL6.

(c) Inhibition of IL6-Dependent Signaling Pathway and Cell Proliferationby Anti-IL6 Antibodies

Human multiple myeloma U266 cells were used to examine the effect ofanti-IL6 antibodies on IL6-induced signaling cascade. U266 cells werecultured in the presence or absent of antibodies, together with rhIL6.After 30 minutes, whole cell lysates were collected and western blotanalysis was performed to examine phosphorylated STAT3 protein(P-STAT3), which is a major transcription factor involved in the IL6signaling pathway. P-STAT3 protein expression was suppressed by the highaffinity anti-IL6 antibodies, Ag1-4-6 and HAg1T-3-10, at a level greaterthan Actemra, an anti-IL6 receptor humanized antibody, was used as acontrol. (FIGS. 1A and 1B). The levels of inhibiting STAT3phorphorylation by the antibodies are shown in Table 3 below:

TABLE 3 Levels of STAT3 phosphorylation inhibition by anti-IL6antibodies inhibition percentage of control in different Abconcentration 0.05 μg/ml 0.5 μg/ml 1-4-62 0 8 Ag1-4-6 0 60 HAg1T-3-10 4993 Actemra 0 17

In addition, to examine the antibody activity on cell proliferation,IL6-dependent B9 murine hybridoma cells were cultured in the presence ofanti-IL6 or anti-IL6R antibodies, as well as an isotype control antibodyat various concentrations. The impact on cell proliferation was assessedby WST-1 assay (Roche) after 72 hours. As shown in FIG. 1C, fully-humananti-IL6 antibodies 1-4-62, Ag1-4-6, and HAg1T-3-10 inhibited B9 cellproliferation in a dose-dependent manner and the IC₅₀ values of 1-4-62,Ag1-4-6, and HAg1T-3-10 are 0.618, 0.0468, and 0.00456 μg/ml,respectively.

Example 3: Effectiveness of High Affinity Anti-IL6 Antibodies in CancerTreatment Materials and Methods (i) Animals

Male NOD/SCID mice (7-8 weeks old) were purchased from BioLASCO TaiwanCo., Ltd (Taipei, Taiwan) for all experiments. The mice were maintainedin sterile individually ventilated cages (IVC) at 20° C. and acclimatedto the housing facilities at least 7 days before experiments wereinitiated.

(ii) Matrigel Angiogenesis Assay

Liquid matrigel (BD Biosciences) was maintained at 4° C. hIL6recombinant protein (R&D Systems) was added to Matrigel to a finalconcentration of 100 ng/0.5 ml. Mice were anesthetized with Avertin (0.2ml/10 g, i.p. injection) and injected at two sites of the dorsal sidewith 0.5 ml of matrigel. Then, the mice were treated with anti-IL6 IgGantibodies or a control IgG antibody through intravenous injection. Onday 6, the mice were euthanized by CO₂ asphyxiation. Matrigel plugs wereremoved, weighed and photographed. To measure hemoglobin levels, theMatrigel plug was lysed with a lysis buffer (1% SDS, 0.5% Triton in PBS)overnight at 4° C. The hemoglobin contents of the plugs were quantitatedusing Drabkin's reagent (Sigma). The concentrations of hemoglobin in theplugs were determined using a standard curve of bovine hemoglobin(Sigma). Hemoglobin contents were indicated as micrograms hemoglobin pergram Matrigel. Mean+SD were calculated using the Student's unpaired ttest; p<0.05 was considered as statistically significant.

(iii) Prostate PC3 Tumor Metastasis Assay Using an IntrasplenicImplantation Model

Mice were anesthetized with Avertin (0.2 ml/10 g) and shaved on the leftflank. They were then placed at the right lateral decubitus position andscrubbed with 75% alcohol at the left flank. The skin at the abdominalwall was incised longitudinally (parallel to the spine) for 1 cm. Thespleen was exteriorized and stabilized gently. A 30-gauge needle on aninsulin syringe was inserted into the parenchyma of the spleen for 3-4mm. 50 μl of a PC-3 cell suspension was injected slowly. A visible palewheal indicates a successful injection. The needle was then retractedand a small cotton ball was placed to cover the injection site for 30seconds to prevent bleeding and spillage of the cell suspension. Thespleen was placed back into the peritoneum. The abdominal wall wasclosed with a 6-0 nylon suture, and the skin was closed with a 4-0 nylonsuture.

After the implantation, the mice were randomly assigned to differenttreatment groups (Docetaxel group Docetaxel plus anti-IL6 antibodygroup, and PBS control group) and were treated with multiple doses ofanti-IL6 antibody (20 mg/kg each time, twice a week), Docetaxel (3 mg/kgeach time, once per week) through the tail vein or by i.p injection. Thebody weights of those mice were measured twice a week.

All mice were then euthanized by CO₂ asphyxiation and body weights weredetermined. Primary tumors in the spleen and liver were excised,photographed, and weighed. Organ index was expressed as milligram organper gram mouse. Mean+SD were calculated using Student's t-test. Thesurvival assay was used Kaplan-Meier analysis. Paraffin-embedded orfrozen section of tumor masses were prepared and stained by anti-CD31and Ki-67 plus Hematoxylin staining for analysis.

(iv) Xenograft Human Pancreatic Tumor Model Human pancreatic tumor cellsBxPC-3 and MiaPaCa cells were re-suspended in PBS and injectedsubcutaneously (s.c.) (5×10⁶ cells, total volume 0.15 mL) into the rightflank of mice. After 10 days, the mice were randomly assigned todifferent treatment groups (Antibody FB704, Oxaliplatin, FB704 plusOxaliplatin, Gemcitabine, and PBS control groups) and treated withmultiple doses of FB704 (20 mg/kg each time, twice a week) orOxaliplatin (3 mg/kg each time, once a week), or Gemcitabine (80 mg/kgeach time, twice per week) through the tail vein or by i.p. injection.Mice body weights and the tumor sizes were measured twice a week. Tumorsizes were measured using a caliper, and the tumor volume was calculatedby length×width×0.52. After the experiment, all mice were sacrificed,mice blood samples were collected and the tumor masses were removed andweighted. The differences in mean tumor volume and tumor weight wereevaluated by Student's t-test. Paraffin-embedded or frozen section oftumor masses were prepared and stained by anti-CD31, Ki67 or TUNEL plusH&E staining for analysis.

Results (i) FB704 Inhibits In Vivo Angiogenesis in a Matrigel Assay

The anti-angiogenic ability of FB704 was examined in an in vivo matrigelmodel as described herein. Six days after the treatment, the mice werescarified mice and hemoglobin concentrations were measured. There was anobservable difference in color and vessel density in excised matrigelplugs of FB704 treated groups (FIG. 4A), indicating the anti-angiogenicactivity of the antibody. FB704 treatment also significantly reduced thehemoglobin concentrations as compared with hIL6. (FIG. 4B).

(ii) FB704 Inhibits Human Prostate Cancer Cell PC-3 Induced Cachexia andMetastasis

To verify the efficacy of FB704 on cancer induced cachexia andmetastasis, mice were implanted with human prostate cancer PC-3 cell(1.0×10⁶ per mouse) by intrasplenic injection. On day 1, the mice wererandomized into 4 groups (n=12-15). High (20 mg/kg) and low (5 mg/kg)dose of FB704, positive control Actemra (20 mg/kg) and PBS wereadministered via i.v. (intravenous) injection twice a week. On day 31after PC-3 cell implantation, the total body weights of PBS treated micewere decreased by approximately 19%. In contrast, the total body weightsof the high dose FB704 and Actemra treated groups remained stable, whichis significant different as compared with the control groups (FIG. 5A;P<0.01).

In addition, after a latency period of days, PC3 tumor-bearing micedeveloped hind leg paralysis or additional symptoms of disseminatedtumor spread. Repetitive intra-vessel injections of FB704 (P=0.0001) andActemra (P=0.024) significantly prolonged symptom-free survival of PC3tumor-bearing mice, wherein FB704 showed significantly better efficacythan Actemra (P=0.03) (FIG. 5B). Gross necropsy of PBS treated groupshowed severe enlargement and tumor cell infiltration in liver. Incontrast, FB704 treated group showed moderate tumor cell infiltrationand more than 50% normal hepatocyte in the left and middle lobes ofliver (FIG. 5C).

Angiogenesis also plays important rule in tumor metastasis.Immunohistochemistry was performed to verify the vessel density on tumorsections. Staining for CD31 (the marker for angiogenesis) showed thatangiogenesis in the FB704 treated group was significantly decreased ascompared with the PBS control group (FIGS. 5D and 5E).

Further, combined treatment of FB704 and chemo-drug Docetaxel providedbetter overall survival rates (FIG. 5F).

(iii) FB704 Enhances the Anti-Tumor Activity of Oxaliplatin orGemcitabine in the Pancreatic Carcinoma Xenograft Model

FB704 enhanced the anti-tumor activity of Oxaliplatin or Gemcitabine ina human pancreatic cancer model. Treatment of BxPC-3 tumor-bearing micewith 20 mg/kg of FB704 twice a week plus Oxaliplatin (3 mg/kg) once aweek and Gemcitabin (80 mg/kg) twice a week resulted in statisticallysignificant tumor growth inhibition of 49% and 60%, respectively(P<0.01) (FIGS. 6A and 6G). The mouse body weight had normally increased(FIG. 6B). The tumor mass weights were significantly reduced after thetreatments (FIGS. 6C and 6D).

An antibody specific to Ki-67, which is a cell proliferation marker, wasused to examine the tumor cell proliferation rates tumor tissues of eachgroup. The percentages of Ki-67 positive cells in the PBS, Oxaliplatin,FB704 and FB704 plus Oxaliplatin treated groups were 26%, 14%, 16% and7.5% respectively (FIGS. 6E and 6F), indicating that the combinedtreatments of FB704 and Oxaliplatin or Gemcitabine provided betterinhibition of pancreatic tumor cell proliferation in vivo.

FB704 also showed synergic benefits with gemcitabine in a pancreaticcancer model. Treatment of BxPC-3 tumor-bearing mice with 20 mg/kg ofFB704 and 80 mg/kg of gemcitabine twice a week resulted in statisticallysignificant tumor growth inhibition (P<0.01) (FIG. 6G).

Example 4: Effectiveness of High Affinity Anti-IL6 Antibodies inRheumatoid Arthritis Treatment

In RA patient, elevated production of chemokine such as MCP-1 has beenobserved in joints, suggesting the involvement of chemokine in thepathogenesis of RA. Adhesion molecule, sICAM-1 also plays importantroles in the infiltration of inflammatory cells into injured tissue.HUVEC naturally expressed sICAM-1 on cell surface. IL6 plus sIL6Rtreatment induced sICAM-1 expression.

(i) Anti-IL6 Antibodies Inhibited MCP-1 and sICAM-1 Production in HUVECCells

To examine the effectiveness of the anti-IL6 antibodies described hereinin treating rheumatoid arthritis (RA), monocyte chemotactic protein-1(MCP-1) and sICAM-1 secretion by Human Umbilical Vein Endothelial Cells(HUVECs) were measured after antibody treatment as follows. The HUVECcells were plated in a 48-well plate at a density of 2×105 cells/ml. Thecells were treated with a combination of recombinant human IL6, IL6receptor-α (R&D systems) with or without anti-IL6 antibodies at variousconcentrations or the control IgG antibody at 37° C. for 24 h. Cell freeculture supernatants were collected and analysis for MCP-1 and sICAM-1by ELISA (RayBiotech)

This in vitro assay indicated that IL6 plus sIL6Ra induced MCP-1 andsICAM-1 expression by HUVECs. Antibodies Ag1-4-6 and HAg1T-3-10suppressed IL6 plus sIL6Ra induced MCP-1 (FIG. 2A) and sICAM-1 (FIG. 2B)in a dose dependent manner. The suppression efficacy was higher thanActemra.

(ii) Anti-IL6 Antibodies Inhibited MCP-1 Production in Both U937 Cellsand Human Peripheral Blood Mononuclear Cell (PBMC)

MCP-1 is a small cytokine that belongs to the CC chemokine family, whichrecruits monocytes, memory T cells, and dendritic cells to the sites ofinflammation. Among immune cells, monocytic cells are known to be themain producers of MCP-1. MCP-1 plays a major role on inflammatory andarthritis. Furthermore, IL6 was shown to induce MCP-1 in humanmonocytes. Therefore, the anti-IL6 antibodies described herein weretested for their effectiveness in suppressing MCP-1 expression in thepromonocytic cell line U937 cells stimulated by IL6.

U937 (2×10⁶ cells/well) were cultured with IL6 (100 ng/ml) for 24 h in a48 flat-bottomed culture plates (Corning, Corning, N.Y.) containingRPMI1640 in the presence of the anti-IL6 antibodies at differentconcentrations. The levels of MCP-1 in the supernatants were measured byMCP-1 ELISA kits (e Bioscience). All of the in vitro experiments wereperformed in triplicate.

The production of MCP-1 from U937 cells was observed after IL6stimulation and in a dose-dependent manner and anti-IL6 antibodiesinhibited the MCP-1 production in U937 cells induced by IL6 (FIG. 7A).

Further, PBMCs were isolated from five healthy donors. The cells (5×10⁵cells/250 μl/well) were cultured with IL6 (100 ng/ml) for 24 h in a96-well U-bottomed culture plates (Corning, Corning, N.Y.) containingRPMI1640 in the presence of anti-IL6 antibodies at differentconcentrations. The levels of MCP-1 in the supernatant were measured byMCP-1 ELISA kits (e Bioscience).

The production of MCP-1 by PBMC cells was observed after IL6stimulation. The presence of the antibodies Ag1-4-6 and HAg1T-3-10inhibited MCP-1 production in a dose dependent manner, while a controlisotype IgG1 did not show this inhibitory effect (FIG. 7B).

(iii) Anti-IL6 Antibodies Inhibited MCP-1 and VEGF Production inSynovial Fibroblasts from Rheumatoid Arthritis Patients

Fresh synovial tissues were minced and digested in a solution ofcollagenase and DNase.

Isolated fibroblasts were filtered through 70-mm nylon filters. Thecells were grown on plastic cell culture dishes in 95% air/5% CO2 inRPMI 1640 (Life Technologies) that was supplemented with 20 mM HEPES and10% heat-inactivated FBS, 2 mM glutamine, 100 Um′ penicillin, and 100mg/ml streptomycin (pH adjusted to 7.6). More than 95% of the cells werefibroblasts, as characterized by immunofluorescence staining using anantibody specific for the fibroblast protein marker vimentin.Fibroblasts from passages four to nine were used for the experiments.

Fibroblast-like synnoviocyte derived from human RA patients as describedabove (RA-FLS cells) were cultured in a 6-well flat-bottomed cultureplates (Corning, Corning, N.Y.; 2×10⁵ cells/2 ml/well) for 2 days andwere stimulated with both IL6 and sIL6R. Antibodies at variousconcentrations were added to the wells and incubated with the cells for24 h. The MCP-1 levels were then measured by MCP-1 ELISA kits (eBioscience).

RA-FLS cells naturally express MCP-1 at low levels and pretreatment withIL6 and sIL6R could stimulate high level MCP-1 production. The anti-IL6antibodies described herein showed significant inhibition of the MCP-1production in commercially available RA-FLS cells (FIG. 8A) and theRA-FLS cells obtained from RA patients as described above (FIG. 8B).

Vascular endothelial growth factor (VEGF) plays an important role in thepathogenesis of RA. VEGF levels are significantly higher in synovialfluids from RA patients. It also induces vascular permeability andmediates inflammation. To examine the effect of anti-IL6 antibodies onVEGF production in RA-FLS cells, those cells were treated with theanti-IL6 antibodies described herein in the presence of IL6 (100 ng/ml),sIL6R (100 ng/ml), and IL1β (5 ng/ml) as follows.

The RA-FLS cells (2×10⁴ cells/500 μl/well) were cultured in a 48-wellflat-bottomed culture plates (Corning, Corning, N.Y.) for 24 hours andthen stimulated with both IL6 and sIL6R and IL-1β. Antibodies at variousconcentrations were added to the wells and incubated with the cells for48 hours. The supernatants were collected and the levels of VEGF weremeasured by Human VEGF ELISA kit (PreproTech).

The VEGF in the culture supernatants of the RA-FLS cells stimulated withthe combination of IL6, IL6R and IL1β was 3-5 fold higher than those inthe supernatants from cells not stimulated by the cytokines. TheAnti-IL6 antibodies significantly reduced Our the VEGF production inboth commercially available RA-FLS cells (FIG. 9A) and the RA-FLS cellsprepared from RA patients as described herein (FIG. 9B).

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

What is claimed is:
 1. An isolated antibody that binds to humaninterleukin 6 (IL6), comprising: (a) a heavy chain variable region(V_(H)), which comprises a heavy chain complementary determining region1 (HC CDR1) of SEQ ID NO: 2, a heavy chain complementary determiningregion 2 (HC CDR2) of SEQ ID NO: 4, and a heavy chain complementarydetermining region 3 (HC CDR3) of SEQ ID NO: 6 or SEQ ID NO: 16; or (b)a light chain variable region (V_(L)), which comprises a light chaincomplementary determining region 1(LC CDR)1 of SEQ ID NO: 9, a lightchain complementary determining region 2 (LC CDR2) of SEQ ID NO: 11, anda light chain complementary determining region 3 (LC CDR3) of SEQ ID NO:13 or SEQ ID NO:
 15. 2. The isolated antibody of claim 1, whichcomprises (i) a V_(H) that comprises the HC CDR1 of SEQ ID NO: 2, the HCCDR2 of SEQ ID NO: 4, and the HC CDR3 of SEQ ID NO: 6; or (ii) a V_(H)that comprises the HC CDR1 of SEQ ID NO: 2, the HC CDR2 of SEQ ID NO: 4,and the HC CDR3 of SEQ ID NO:
 16. 3. The isolated antibody of claim 2,which comprises a V_(H) that comprises the amino acid sequence of SEQ IDNO:17 or SEQ ID NO:18.
 4. The isolated antibody of claim 2 or claim 3,which further comprises (i) a V_(L) that comprises the LC CDR1 of SEQ IDNO: 9, the LC CDR2 of SEQ ID NO: 11, and the LC CDR3 of SEQ ID NO: 13;or (ii) a V_(L) that comprises the LC CDR1 of SEQ ID NO: 9, the LC CDR2of SEQ ID NO: 11, and the LC CDR3 of SEQ ID NO:
 15. 5. The isolatedantibody of claim 4, which comprises a V_(L) that comprises the aminoacid sequence of SEQ ID NO:19 or SEQ ID NO:20.
 6. The isolated antibodyof claim 1, which is selected from the group consisting of: (i) anantibody comprising a V_(H) that comprises the HC CDR1 of SEQ ID NO: 2,the HC CDR2 of SEQ ID NO: 4, and the HC CDR3 of SEQ ID NO: 6; and aV_(L) that comprises the LC CDR1 of SEQ ID NO: 9, the LC CDR2 of SEQ IDNO: 11, and the CDR3 of SEQ ID NO: 13; (ii) an antibody comprising aV_(H) that comprises the HC CDR1 of SEQ ID NO: 2, the HC CDR2 of SEQ IDNO: 4, and the HC CDR3 of SEQ ID NO: 16, and a V_(L) that comprises theLC CDR1 of SEQ ID NO: 9, the LC CDR2 of SEQ ID NO: 11, and the CDR3 ofSEQ ID NO: 13; (iii) an antibody comprising a V_(H) that comprises theHC CDR1 of SEQ ID NO: 2, the HC CDR2 of SEQ ID NO: 4, and the HC CDR3 ofSEQ ID NO: 6, and a V_(L) that comprises the LC CDR1 of SEQ ID NO: 9,the LC CDR2 of SEQ ID NO: 11, and the CDR3 of SEQ ID NO: 15; and (iv) anantibody comprising a V_(H) that comprises the HC CDR1 of SEQ ID NO: 2,the HC CDR2 of SEQ ID NO: 4, and the HC CDR3 of SEQ ID NO: 16, and aV_(L) that comprises the LC CDR1 of SEQ ID NO: 9, the LC CDR2 of SEQ IDNO: 11, and the CDR3 of SEQ ID NO:
 15. 7. The isolated antibody of claim6, which is selected from the group consisting of: (i) an antibodycomprising a V_(H) that comprises the amino acid sequence of SEQ ID NO:17, and a V_(L) that comprises the amino acid sequence of SEQ ID NO:19;(ii) an antibody comprising a V_(H) that comprises the amino acidsequence of SEQ ID NO: 17, and a V_(L) that comprises the amino acidsequence of SEQ ID NO:20; and (iii) an antibody comprising a V_(H) thatcomprises the amino acid sequence of SEQ ID NO: 18, and a V_(L) thatcomprises the amino acid sequence of SEQ ID NO:19; and (iv) an antibodycomprising a V_(H) that comprises the amino acid sequence of SEQ ID NO:18, and a V_(L) that comprises the amino acid sequence of SEQ ID NO:20.8. The isolated antibody of any of claims 1-7, wherein the antibody is afull-length antibody or an antigen-binding fragment thereof.
 9. Theisolated antibody of claim 8, wherein the antigen-binding fragmentthereof is Fab or (Fab′)₂.
 10. The isolated antibody of any of claims1-8, wherein the antibody is a single chain antibody.
 11. The isolatedantibody of any of claims 1-8, wherein the antibody is a humanizedantibody or a human antibody.
 12. A composition comprising an antibodyof any of claims 1-11 and a carrier.
 13. The composition of claim 12,wherein the composition is a pharmaceutical composition that comprises apharmaceutically acceptable carrier.
 14. A combination comprising anantibody of any of claims 1-11 and another anti-cancer agent or adisease modifying antirheumatic drug (DMARD).
 15. The combination ofclaim 14, wherein the anti-cancer agent is selected from the groupconsisting of docetaxel, oxaliplatin, and gemcitabine.
 16. Thecombination of claim 14, wherein the DMARDs is selected from the groupconsisting of methotrexate, azathioprine, chloroquinehydroxychloroquine, cyclosporin A, and sulfasalazine.
 17. An antibodyfor use in medicament, wherein the antibody is any of claims 1-11.
 18. Apharmaceutical composition for use in treating a disease associated withIL6, wherein the pharmaceutical composition comprises an antibody of anyof claims 1-11 and a pharmaceutically acceptable carrier.
 19. Thepharmaceutical composition for use of claim 18, wherein the diseaseassociated with IL6 is selected from the group consisting ofinflammatory disorder, autoimmune diseases, which optionally is selectedfrom the group consisting of rheumatoid arthritis, Crohn's disease,Castleman's disease, multiple sclerosis, ankylosing spondylitis,psoriatic arthritis and psoriasis, angiogenesis, cancer, which isoptionally selected from the group consisting of multiple myeloma,leukemia, breast cancer, pancreatic cancer, lung cancer, ovarian cancer,oral cancer and prostate cancer, tumor metastasis, cancer relatedcachexia.
 20. The pharmaceutical composition for use of claim 18,wherein the disease associated with IL6 is cancer.
 21. Thepharmaceutical composition for use of claim 18, wherein thepharmaceutical composition is co-used with another anti-cancer agent.22. The pharmaceutical composition for use of claim 21, wherein theanti-cancer agent is selected from the group consisting of oxaliplatin,gemcitabine, and docetaxel.
 23. The pharmaceutical composition for useof claim 18, wherein the disease associated with IL6 is an autoimmunedisease.
 24. The pharmaceutical composition for use of claim 23, whereinthe autoimmune disease is RA.
 25. The pharmaceutical composition for useof claim 24, wherein the pharmaceutical composition is co-used with oneor more disease modifying antirheumatic drugs (DMARDs).
 26. Thepharmaceutical composition for use of claim 25, wherein the DMARDs isselected from the group consisting of methotrexate, azathioprine,chloroquine hydroxychloroquine, cyclosporin A, and sulfasalazine.
 27. Amethod for treating a disease associated with IL6, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of an antibody of any of claims 1-11.
 28. The method of claim 27,wherein the disease associated with IL6 is selected from the groupconsisting of inflammatory disorder, autoimmune diseases, angiogenesis,and cancer.
 29. The method of claim 28, wherein the disease associatedwith IL6 is cancer, which is selected from the group consisting ofmultiple myeloma, leukemia, breast cancer, pancreatic cancer, lungcancer, ovarian cancer, oral cancer and prostate cancer.
 30. The methodof claim 29, wherein the amount of the antibody is effective in reducingtumor metastasis or cancer related cachexia.
 31. The method of claim 28or claim 29, further comprising administering to the subject anotheranti-cancer agent.
 32. The method of claim 31, wherein the otheranti-cancer agent is selected from the group consisting of oxaliplatin,gemcitabine, and docetaxel.
 33. The method of claim 28, wherein thedisease associated with IL6 is an autoimmune disease selected from thegroup consisting of rheumatoid arthritis (RA), Crohn's disease,Castleman's disease, multiple sclerosis, ankylosing spondylitis,psoriatic arthritis and psoriasis.
 34. The method of claim 33, furthercomprising administering to the subject one or more disease modifyingantirheumatic drugs (DMARDs).
 35. The method of claim 34, wherein theDMARD is selected from the group consisting of methotrexate,azathioprine, chloroquine, hydroxychloroquine, cyclosporin A,sulfasalazine.
 36. Use of the antibody of any of claims 1-11 in themanufacture of a medicament for treating a disease or conditionassociated with IL6.
 37. A nucleic acid comprising a nucleotide sequenceencoding an antibody heavy chain variable region (V_(H)), an antibodylight chain variable region (V_(L)) or both, wherein the V_(H) and V_(L)is set forth in any of claims 1-7.
 38. A vector comprising the nucleicacid of claim
 37. 39. The vector of claim 38, wherein the vector is anexpression vector.
 40. A host cell comprising the nucleic acid of claim37 or the vector of claims 38 or
 39. 41. A method for producing anantibody that binds to human IL6, comprising: culturing the host cell ofclaim 40 under conditions allowing for expression of the antibody. 42.The method of claim 41, further comprising harvesting the antibody.